The present invention relates generally to cell surface receptors, and more specifically, to Corticotropin-Releasing Factor2 receptors.
Corticotropin-releasing factor (xe2x80x9cCRFxe2x80x9d) is a 41-amino acid peptide originally isolated from the hypothalamus by virtue of its ability to stimulate the production of adrenocorticotropic hormone (xe2x80x9cACTHxe2x80x9d) and other proopiomelanocortin (xe2x80x9cPOMCxe2x80x9d) products of the anterior pituitary (Vale et al., Science 213:1394-1397, 1981). Briefly, CRF is believed to initiate its biological effects by binding to a plasma membrane receptor which is distributed throughout the brain (DeSouza et al., Science 224:1449-1451, 1984), pituitary (Wynn et al., Biochem. Biophys. Res. Comm. 110:602-608, 1983), adrenals (Udelsman et al., Nature 319:147-150, 1986) and spleen (Webster, E. L., and E. B. DeSouza, Endocrinology 122:609-617, 1988). This receptor is coupled to a GTP-binding protein (Perrin et al., Endocrinology 118:1171-1179, 1986) which mediates CRF-stimulated increase in intracellular cAMP (Bilezikjian, L. M., and W. W. Vale, Endocrinology 113:657-662, 1983).
In addition to its role in stimulating the production of ACTH and POMC, CRF is also believed to coordinate many of the endocrine, autonomic, and behavioral responses to stress, and may be involved in the pathophysiology of affective disorders. Moreover, CRF is believed to be a key intermediary in communication between the immune, central nervous, endocrine and cardiovascular systems (Crofford et al., J. Clin. Invest. 90:2555-2564, 1992; Sapolsky et al., Science 238:522-524, 1987; Tilders et al., Regul. Peptides 5:77-84, 1982; Fisher et al., Reg. Peptide 5:153-161, 1983).
A receptor for CRF has been cloned from rat (Perrin et al., Endo 133(6):3058-3061, 1993), and human brain (Chen et al., PNAS 90(19):8967-8971, 1993; Vita et al., FEBS 335(1):1-5, 1993). This receptor is a 415 amino acid protein comprising seven membrane spanning domains, and has a predicted molecular weight of 44,000 daltons. A comparison of identity between rat and human sequences shows a high degree of homology (97%) at the amino acid level. In addition, Scatchard analysis of recombinantly produced human receptor demonstrates a single component site, with a high affinity (Kd of 1.6xc2x10.3 nM) for CRF.
The present invention provides new, previously unidentified CRF receptors, designated as the xe2x80x9cCorticotropin-Releasing Factor-2 (xe2x80x9cCRF2xe2x80x9d), Corticotropin-Releasing Factor receptors. In addition, the present invention provides compositions and methods which utilize such CRF2 receptors, as well as other, related advantages.
Briefly stated, the present invention provides compositions and methods which utilize CRF2 receptors (also termed xe2x80x9cCRF2 Corticotropin-Releasing Factor receptorsxe2x80x9d or xe2x80x9cCRF2Rxe2x80x9d). Within one aspect of the present invention, isolated nucleic acid molecules are provided which encode CRF2 receptors. Within one embodiment, nucleic acid molecules are provided which encode a CRF2 receptor such as that disclosed in Sequence I.D. No. 4, from amino acid number 1 to amino acid number 411. Within another embodiment, nucleic acid molecules are provided which comprise the sequence of nucleotides in Sequence I.D. No. 3, from nucleotide number 216 to nucleotide number 1449. Within another embodiment, nucleic acid molecules are provided which encode a CRF2 receptor such as that disclosed in Sequence ID No. 2 from amino acid number 1 to amino acid number 431. Within another embodiment, nucleic acid molecules are provided which comprise the sequence of nucleotides in Sequence ID No. 1 from nucleotide number 44 to nucleotide number 1336. Nucleic acid molecules which encode CRF2 receptors of the present invention may be isolated from virtually any warm-blooded animal, including for example, humans, macaques, horses, cattle, sheep, pigs, dogs, cats, rats and mice.
Within another aspect of the present invention, isolated nucleic acid molecules are provided which encode portions of a CRF2 receptor, such as the N-terminal extracellular domain. Within one embodiment, isolated nucleic acid molecules are provided comprising the sequence of nucleotides in Sequence ID. No. 3, from nucleotide number 216 to nucleotide number 570. Within another embodiment, isolated nucleic acid molecules are provided which encode a protein having the amino acid sequence of Sequence I.D. No. 4, from amino acid number 1 to amino acid number 118. Within another embodiment, isolated nucleic acid molecules are provided comprising the sequence of nucleotides in Sequence ID No. 1 from nucleotide number 44 to nucleotide number 451. Within another embodiment, isolated nucleic acid molecules are provided which encode a protein having the amino acid sequence of Sequence ID No. 2 from amino acid number 1 to amino acid number 138.
Within other aspects of the invention, expression vectors are provided which are capable of expressing the above-described nucleic acid molecules. Within other aspects, recombinant viral vectors are provided which are capable of directing the expression of the above-described nucleic acid molecules. Representative examples of such viral vectors include retroviral vectors, adenoviral vectors, and herpes simplex virus vectors. Also provided by the present invention are host cells which contain the above-described expression vectors, as well as the receptor or portions thereof which are encoded by the above-described nucleic acid molecules. Within other embodiments, isolated portions of CRF2 receptors are provided, including for example, isolated portions of extracellular domains such as the N-terminal extracellular domain.
Within other aspects of the invention, isolated antibodies are provided which are capable of specifically binding to the above-described CRF2 receptors. Within one embodiment, the antibodies may be selected from the group consisting of polyclonal antibodies, monoclonal antibodies, and antibody fragments. Within other embodiments, antibodies are provided which are capable of blocking the binding of CRF (or other substrates such as sauvagine or urotensin I) to a CRF2 receptor. Within preferred embodiments, the antibodies may be selected from the group consisting of murine and human antibodies. Within preferred aspects of the invention, the above-noted antibodies are produced by hybridomas.
Within yet another aspect of the present invention, nucleic acid molecules are provided which are capable of specifically hybridizing to a nucleic acid molecule encoding any of the CRF2 receptors described above. Such molecules may be between at least xe2x80x9cyxe2x80x9d nucleotides long, wherein xe2x80x9cyxe2x80x9d is any integer between 14 and 1230, and furthermore, may be selected suitable for use as probes or primers described below. Particularly preferred probes of the present invention are at least 18 nucleotides in length.
Within other aspects of the present invention, methods for detecting the presence of a compound which binds to a CRF2 receptor are provided, comprising the steps of (a) exposing one or more compounds to cells that express CRF2 receptors under conditions and for a time sufficient to allow binding of the compounds to the receptors, and (b) isolating compounds which bind to the receptors, such that the presence of a compound which binds to a CRF2 receptor may be detected. Within another aspect, methods for detecting the presence of a compound which binds to a CRF2 receptor are provided, comprising the steps of (a) exposing one or more compounds to a CRF2 receptor N-terminal extracellular domain under conditions and for a time sufficient to allow binding of a compound to the N-terminal extracellular domain, and (b) isolating compounds which bind to the CRF2 receptor N-terminal extracellular domain, such that the presence of a compound which binds to a CRF2 receptor may be detected. Within one embodiment, the compounds are labeled with an agent selected from the group consisting of fluorescent molecules, enzymes, and radionuclides.
Within other aspects of the present invention, methods for determining whether a selected compound is a CRF2 receptor agonist or antagonist are provided, comprising the steps of (a) exposing a selected compound to cells which express CRF2 receptors under conditions and for a time sufficient to allow binding of the compound and an associated response in intracellular levels of cAMP, and (b) detecting either an increase or decrease in the level of intracellular cAMP, and thereby determining whether the selected compound is a CRF2 receptor agonist or antagonist.
Within other aspects methods are provided for detecting the presence of a CRF2 receptor agonist or antagonist in a pool of compounds, comprising the steps of (a) exposing a pool of compounds to cells which express CRF2 receptors under conditions and for a time sufficient to allow binding of the compound and an associated response in intracellular levels of cAMP, and (b) isolating compounds which either increase or decrease the intracellular level of cAMP, such that the presence of a CRF2 receptor agonist or antagonist may be detected.
Within another aspect, methods for determining whether a selected compound is a CRF2 receptor antagonist are provided, comprising the steps of (a) exposing a selected compound in the presence of a CRF2 receptor agonist to a recombinant CRF2 receptor coupled to a response pathway under conditions and for a time sufficient to allow binding of the compound to the receptor and an associated response through the pathway, and (b) detecting a reduction in the stimulation of the response pathway resulting from the binding of the compound to the CRF2 receptor, relative to the stimulation of the response pathway by the CRF2 receptor agonist alone. and therefrom determining the presence of a CRF2 antagonist. Within other aspects, methods are provided for determining whether a selected compound is a CRF2 receptor agonist, comprising the steps of (a) exposing a selected compound to a recombinant CRF2 receptor coupled to a response pathway under conditions and for a time sufficient to allow binding of the compound to the receptor and an associated response through the pathway, and (b) detecting an increase in stimulation of the response pathway resulting from the binding of the compound to the CRF2 receptor, and therefrom determining the presence of a CRF2 receptor agonist.
Within other aspects of the present invention, methods are provided for treating CRF2 receptor-associated diseases, wherein it is desired to either increase or decrease stimulation of a CRF2 receptor response pathway. For example, within one aspect methods are provided for treating cerebrovascular disorders such as stroke, reperfusion injury and migraines, comprising the step of administering to a patient a therapeutically effective amount of a CRF2 receptor antagonist, such that the disorder is remedied or alleviated. Within other aspects, methods are provided for treating learning or memory disorders, comprising administering to a patient a therapeutically effective amount of a CRF2 receptor antagonist. Within yet other aspects, methods are provided for treating Alzheimer disease, comprising administering to a patient a therapeutically effective amount of a CRF2 receptor antagonist. Representative examples of suitable CRF2 receptor antagonist include xcex1-helical oCRF (9-41), or d-Phe r/h CRF (12-41).
These and other aspects of the present invention will become evident upon reference to the following detailed description and attached drawings. In addition, various references are set forth below which describe in more detail certain procedures or compositions (e.g., plasmids, etc.), and are therefore incorporated by reference in their entirety.